1. Field of the Invention
The present invention relates to the control of gene expression and, in particular, it relates to methods of, and means for, suppressing the expression of a particular, selected gene.
2. Related Art
The ability to selectively suppress the expression of a gene is useful in many areas of biology, for example in methods of treatment where the expression of the gene may be undesirable; in preparing models of disease where lack of expression of a particular gene is associated with the disease; in modifying the phenotype in order to produce desirable properties. Thus, the ability to selectively suppress the expression of a gene may allow the “knockout” of human genes in human cells (whether wild type or mutant) and the knockout of eukaryotic genes in studies of development and differentiation.
Present methods of attempting to suppress the expression of a particular gene fall into three main categories, namely antisense technology, ribozyme technology and targeted gene deletion brought about by homologous recombination.
Antisense techniques rely on the introduction of a nucleic acid molecule into a cell which typically is complementary to a mRNA expressed by the selected gene. The antisense molecule typically suppresses translation of the mRNA molecule and prevents the expression of the polypeptide encoded by the gene. Modifications of the antisense technique may prevent the transcription of the selected gene by the antisense molecule binding to the gene's DNA to form a triple helix.
Ribozyme techniques rely on the introduction of a nucleic acid molecule into a cell which expresses a RNA molecule which binds to, and catalyses the selective cleavage of, a target RNA molecule. The target RNA molecule is typically a mRNA molecule, but it may be, for example, a retroviral RNA molecule.
Antisense- and ribozyme-based techniques have proven difficult to implement and they show varying degrees of success in target gene suppression or inactivation. Furthermore, these two techniques require persistent expression or administration of the gene-inactivating agent.
Targeted gene deletion by homologous recombination requires two gene-inactivating events (one for each allele) and is not easily applicable to primary cells, particularly for example primary human mammary cells which can only be maintained in culture for a few passages. Targeted gene deletion has remained difficult to perform in plants. The cre-lox mediated site-specific integration has been the method of choice although the efficiency of specific integrative events is low (Alberts et al (1995) Plant J. 7, 649–659; Vergunst & Hooykass (1998) Plant Mol. Biol. 38, 393–406; Vergunst et al (1998) Nucl. Acids Res. 26, 2729–2734).
These major shortcomings in existing technology have led us to seek an alternative strategy.
Acute promyelocytic leukaemia (APL) is underlined by the involvement of mutant retinoic acid receptor (RAR) proteins, formed by gene fusions brought about by chromosomal translocations. Molecular analysis of one APL subset has identified a fusion between the RARα gene and a Kruppel-like zinc finger gene named promyelocytic leukaemia zinc finger (PLZF). Further investigations have shown that the resulting PLZF-RARα fusion protein functions as a gene repressor by targeting histone deacetylation of retinoic acid regulated genes. Several studies have shown that this repression is mediated by the PLZF portion of the fusion protein, which interacts with a complex of proteins which includes the components N-CoR, SMRT, Sin3 and HDAC and which in turn results in the recruitment of the histone deacetylase (HDAC) complex to target genes (see, for example, Grignani et al (1998) Nature 391, 815–818; Chen et al (1993) EMBO J. 12, 1161–1167; Razin (1998) EMBO J. 17, 4905–4908; David et al (1998) Oncogene 16, 2549–2556; and Lin et al (1998) Nature 391, 811–814). HDAC directed gene inactivation, therefore results from the targeted assembly of components, some of which have been identified (eg N-CoR, SMRT, Sin3 etc) making a gene inactivating complex which mediates its effect through histone deacetylation.
Although this work shows that in certain forms of APL fusion proteins are able to recruit histone deacetylase activity which appears to have the effect of inactivating the expression of certain genes, no-one has suggested that a method can be devised based on recruitment of histone deacetylase or other means of inactivating chromatin in order to selectively suppress expression of a chosen target gene or a set of genes. Surprisingly, we have shown that this can be achieved.
RARα-PLZF and RARα-PML fusion proteins are known from studies of acute promyelocytic leukaemia (APL) and are described in, for example, Grignani et al (1998) Nature 391, 815–818.
Fusions of GAL4 with a portion of PLZF protein, and LexA DNA binding domain fused to various fragment of Sin 3A are described in David et al (1998) Oncogene 16, 2549–2556 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention. Fusions of the GAL4 DNA binding domain and PLZF-RARα are described in Lin et al (1998) Nature 391, 811–814 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention.
Fusions of the GAL4 DNA binding domain with N-CoR or portions thereof, or with the C terminal domain of the T3Rβ1 receptor molecule (thyroid hormone receptor molecule), and LexA DNA binding domain fused with the C terminal domain of the T3Rα or RARα (retinoic acid receptor) receptor molecules, which, for the avoidance of doubt, are excluded from the polypeptide of the present invention, are described in Hörlein et al (1995) Nature 377, 397404. Fusions of the GAL4 DNA binding domain with the C terminal domain of vErbA (viral oncogene erbA of the avian erythroblastosis virus (AEV)), T3R and RAR receptor molecules are also mentioned. These polypeptides are also, for the avoidance of doubt, excluded from the polypeptide of the present invention.
There is no suggestion in David et al (1998) Oncogene 16, 2549–2556, Lin et al (1998) Nature 391, 811–814 or Hörlein et al (1995) Nature 377, 397–404 that polypeptides comprising a nucleic acid binding portion and a chromatin inactivation portion can be designed and engineered to bring about the selective suppression of a chosen gene. Rather, David et al (1998) and Lin et al (1998) are both studies of gene repression in acute promyelocytic leukaemia, and Hörlein et al (1995) relates to the identification of N-CoR.